Chronic sepsis is associated with profound muscle wasting. Studies conducted during the current project period have established that sepsis causes an inhibition of protein synthesis in skeletal muscles composed of fast-twitch fibers but not in muscles composed of slow-twitch fibers. Furthermore, the studies have provided evidence that the inhibition of protein synthesis is due to a defect in peptide-chain initiation rather that the result of alterations in cellular RNA content or peptide-chain elongation. The defect in peptide-chain initiation has been localized to a loss of eukaryotic initiation factor eIF-2B activity. The decreased eIF- 2B activity does not result from increased phosphorylation of another initiation factor eIF-2, or changes in allosteric regulators (sugar phosphates; NADH/NAD+; or NADPH/NADP+) of eIF-2B. Instead, preliminary studies have provided evidence that the content of the epsilon-subunit of eIF-2B (eIF-2Bepsilon) in gastrocnemius reduced by 40% in septic rats compared to controls. The hypothesis to be examined in the proposed studies is that an important cause of the sepsis-induced inhibition of peptide-chain initiation, and hence protein synthesis, in fast-twitch muscles of septic rats is the loss of eIF-2B activity. Decreased eIF-2B would be expected to limit the formation of 43S preinitiation complex formation, leading to an inhibition of peptide-chain initiation. The specific aims of the studies proposed for the next project period are; (1) to define the temporal relationship between change s in the expression of the five individual subunits of eIF-2B and the rate of protein synthesis in gastrocnemius, psoas, and heart following induction of a sterile or septic abscess in rats: (2) to investigate factors influencing the expression of the subunits of eIF-2B in sepsis by determining the relative abundance and/or translational efficiency of mRNA for the individual subunits of the factor: (3) to evaluate the effect of sepsis on the extent of phosphorylation of eIF-2Bepsilon; (4) to measure 43S preinitiation complex formation in skeletal muscle composed of fast-twitch fibers during sepsis; and (5) to establish the role of IL-1beta in mediating the changes in eIF- 2B protein expression and/or phosphorylation state during sepsis. We will compare and contrast the effects of sepsis on the regulation of protein metabolism in muscles composed of fast-twitch fibers with muscles composed of slow-twitch fibers. Chronic, hemodynamically stable sepsis will be induced by the creation of an intra-abdominal abscess using an E. coli plus B. fragilis infected sterile fecalagar pellet as the foreign body nidus. The studies will utilize a number of methodologies including in vivo and in vitro measurement of protein synthesis; isolation, purification, immunochemical identification and quantification of specific proteins and mRNA; and estimation of the extent protein phosphorylation. The proposed studies will identify the biochemical locus and mechanism responsible for the inhibition peptide-chain initiation and, hence, protein synthesis in sepsis.